Acquired immunodeficiency syndrome (AIDS), the pandemic infection caused by human immunodeficiency virus-1 (HIV-1), has created an urgent need for new classes of antiviral agents. HIV-1 has infected over 60 million and killed over 20 million individuals worldwide since the beginning of the epidemic (WHO/UNAIDS, December 2005, AIDS Epidemic Update).
HIV infection is not curable. To date, there is no HIV vaccine. There are currently four classes of therapeutics for HIV treatment: nucleoside reverse transcriptase inhibitors, non-nucleoside transcriptase inhibitors, protease inhibitors and fusion inhibitors. Fusion inhibition, which blocks interaction of virus with either or both host cell receptors, is considered to be one of the most effective approaches to prevent and inhibit viral infections. To date, very few fusion inhibitors have been identified.
The primary targets for HIV-1 infection in vivo are CD4+ T cells and cells of the monocyte/macrophage lineage (Klatzmann et al., 1984, Nature 312: 767-8; Dalgleish et al., 1984, Nature 312: 763-7). The initial, critical step of HW infection is its cell entry through the fusion of the viral membrane with the membrane of either a T-cell or macrophage. Major advances have been made over the past decade in the understanding of the molecular machinery of HIV entry into these target cells. An initial step in the entry process is the interaction of the external HIV envelope glycoprotein, gp120, with T-cell CD4 receptor molecules. The functional HIV-1 envelope complex is a trimeric structure comprising three gp120 surface glycoproteins, each noncovalently attached to one of three subunits of the gp41 transmembrane glycoproteins (Chan et al., 1997, Cell 89: 263-73; Wyatt et al., 1998, Science 280: 1884-8; Tan et al., 1997, Proc Natl Acad Sci USA 94: 12303-8). Recent crystal structures of gp120-CD4 with co-receptor surrogate antibody complexes have provided insights into the formation of protein-protein interactions in the process of viral entry (Kwong et al., 1998, Nature 393: 648-59; Huang et al., 2005, Structure 13: 755-68; Huang et al., 2005, Science 310: 1025-8). The binding of gp120 to CD4 receptor promotes a conformational rearrangement in the envelope gp120, that creates a new site for binding of another co-receptor, CCR5 or CXCR4 (Wu et al, 1996, Nature 384: 179-83; Dragic et al., 1996, Nature 381: 667-73). The interaction of virus envelope gp120-CD4 complex with co-receptor is believed to promote further conformational rearrangements in HIV-1 envelope that drive fusion of the viral and host cell membranes. Blocking the binding of CD4 with gp120 or preventing the CD4-induced conformational isomerization that promotes co-receptor binding and viral cell fusion are believed to have great potential for the prevention and treatment of HIV-1 infection and AIDS.
Currently, the development of effective HIV entry inhibitors are mainly focused on natural ligands (Doranz et al., 1997, Immunol Res 16: 15-28; Munk et al., 2003, AIDS Res Hum Retroviruses 19: 875-81), monoclonal antibodies (Gallo et al., 2006, J Biol Chem 281: 18787-92; Zhang et al., 2007, Curr Pharm Des 13: 203-12; Cardoso et al., 2005, Immunity 22: 163-73; Zhang et al., 2003, J Immunol Methods 283: 17-25), and small synthetic compounds, obtained either by high-throughput screening of large compound libraries (Lin et al., 2003, Proc Natl Acad Sci USA 100: 11013-8; Zhao et al., 2005, Virology 339: 213-25; Ferrer et al., 1999, J Virol 73: 5795-802) or structure-guided rationally-designed compounds that interfere with gp120/CD4 or co-receptor interaction (Vita et al., 1999, Proc Natl Acad Sci USA 96: 13091-6; DeMarco et al., 2006, Bioorg Med Chem 14: 8396-404).
Recent investigations using both in vitro and in vivo assays have demonstrated the potential topical microbiocide activity of cyanovirin-N (CV-N), an 11 kD protein originally isolated from the cyanobacteria Nostoc ellipsosporum (Boyd et al., 1997, Antimicro Agents Chemother. 41:1521-1530), CV-N inactivates a broad range of M-tropic and T-tropic strains of HIV-1, SIV, FIV and prevents cell-to-cell transmission of infection (Boyd et al., 1997, Antimicro Agents Chemother. 41; 1521-1530). CV-N binds specifically to the highly glycosylated viral envelope protein gp120 and to the functionally analogous SIV proteins sgp130 and sgp140. The epitopes on gp120 responsible for CV-N binding appear to be predominantly high-mannose glycosylation sites of the envelope. Recombinant CV-N blocks HIV-1 BaL infection of human ectocervical explants without cytotoxic effects (Tsai et al., 2004, AIDS Res Hum Retroviruses 20:11-18). Gel formulations of CVN applied rectally to male macaques protected against challenge by the SIV/HIV-1 virus SHIV89.6P (Tsai et al, 2003, AIDS Res Hum Retroviruses 19:535-541). In vivo efficacy has also be shown in a vaginal challenge model with female macaques (Tsai et al., 2004, AIDS Res Hum Retroviruses 20:11-18). CV-N showed no clinically adverse effects in these in vivo assays. However, the production costs and consequent cost per dose are limitations of the usage of CV-N alone as a therapeutic.
A screen of a random peptide phage-display library identified several peptides that bind to HIV-1 envelope glycoprotein gp120 (Ferrer et al., (1999, Virol. 73:5795-5802). One 12-mer, named 12p1, was found to inhibit the interaction between gp120 and four-domain soluble CD4 (4dCD4) and between gp120 and 17b, an HIV neutralizing monoclonal antibody. Recently, a derivative of 12p1, named HNG-105, obtained using a stable and chemically accessible azidoproline residue as a basis for side-chain bioconjugation reactions through click chemistry has been reported (U.S. Pat. Publication No. 20060135746; Gopi et al., 2006, ChemMedChem 1:54-57). Specifically, proline 6 of the 12p1 peptide was replaced with (2S,4S)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylic acid. The resulting derivative, HNG-105, has a greater binding affinity for gp120, compared to 12p1, and also inhibits strongly the interaction between gp120 and both CD4 and 17b. Furthermore, HNG-105 showed inhibitory effects over a wide range of HIV-1 clades (Cocklin et al., 2007, J Viol, 81:3645-3648). HNG-105 inhibited viral infection with IC50 values ranging from about 105 nM to about 865 nM.
There are currently over twenty medications approved for HIV-1 treatment, only two of which are fusion inhibitors. The development of drug resistant HIV is an on-going problem. Thus, there is a need for new HIV therapeutics. This invention addresses this need.